Study On The Methods For Detecting The Sperm Quality Of Pinctada Martensii

In this paper, the semen of Pinctada martensii was used as the model for studying its viability rate, membrane and DNA integrity with tyban blue and fluorescent staining through a microscope. These can offer basic data for building its sperm quality detection and evaluation.1. Study on sperms viability rate of P. martensii with typan blue stainingThe sperms of P. martensii were stained with typan blue, which appeared obvious difference between the dead and live sperms. The live sperms were not stained, while the dead ones were stained by typan blue and the size of dead ones was bigger than the lives. Then we compare different condition dye concentration and dying time, the best condition was that 0.2 % trypan blue under staining 15 min.Then, the sperms were killed at 60℃(water bath )were mixed the fresh ones to obtain different rations (for dead sperms) concentrations(10 %,20 %,30 %,40 %,50 %,60 %,70 %,80 %,90 %) ,which were identified as theoretical mortality and detected with 0.2 % typan blue and 0.04 % esion Y. The results showed that there was significant correlation between theoretical mortality and practical mortality (P<0.01), and there was a positive correlation between two staining methods (P<0.01).Finally, we made a trial about the toxicity of dye and the natural mortality at room temperature (28℃).The result indicated that the toxicity of these dye will affect the viability rate of sperms when they reached a certain level. But, there was no remarkable toxic effect on viability rate of sperms within 45 min.In conclusion, the optimum dye concentration was 0.2 % and staining time was 15 min. Tyban blue staining was a valuable tool to assessing the sperms of P. martensii. 2. Reseacrh on sperms viability rate and membrane integrity of P. martensii with a combination of fluorometric stainingWe investigated the combination of Fluorescein diacetate (FDA) and propidium iodide (PI) under fluorescence microscope to study sperm viability rate and sperms membrane integrity. The sperms were stained with FDA and PI, which appeared clear difference between the dead and live sperms: the live sperms were green and the dead ones were red under the fluorescent microscopy. Comparing the different staining time and dye concentration, we found that final staining concentration of FDA and PI was 10 ug/mL and the optimum staining time was 10 min.Then, these sperms were killed at 60℃(water bath) were mixed with fresh ones so as to obtain six rations (for dead sperms) concentrations (0 %, 20 %, 40 %, 60 %, 80 %), which were referred to as theoretical mortality and detected with FDA and PI staining. There was a correlation did exist between theoretical and practical mortality of sperms (P<0.01). And there were no difference among the different adding orders of FDA and PI (P<0.01).We evaluate the effect of freezing-thawing process on sperms viability and membrane integrity with FDA-PI staining. The result showed that the mortality highly increased after freezing-thawing (P<0.01). The freezing-thawing process significantly reduced sperms viability and inordinately damages the membrane integrity.Finally, we compared the effect of mortality with different volume after freezing-thawing. The result showed that the volume was too large or too small will affect the sperms of viability and membrane integrity and the optimum volume was 1.2-1.6 mL.In conclusion, the dual association of FDA-PI staining possesses high sensitivity, good repeatability and accuracy. The combination of FDA and PI can be an effective tool for assessing the viability of P. martensii sperm in the future. 3. Study on sperms DNA integrity of P. martensii with Acridine orange (AO) stainingSperm DNA integrity was analyzed using fluorescein staining of AO. The double-stranded DNA were green and single-stranded DNA were red or yellow-orange under the fluorescent microscopy. Comparing the different staining time and dye concentration, we found that final staining concentration of AO was 200ug/mL and the optimum staining time was 5 min. Then, we evaluate the effect of freezing-thawing process on sperm DNA integrity using AO staining. The result showed that the integrity of DNA highly lowered after freezing-thawing (P<0.01). Freezing-thawing process significantly lowered and inordinately damages the double stranded structure of sperm DNA.